The EMBO Journal vol.10 no.2 pp.269-275, 1991

Distinct functions for thyroid hormone receptors ox and ,B in brain development indicated by differential expression of receptor genes Douglas Forrest, Finn Hallbook', Hhkan Persson1 and Bjorn Vennstrom Department of Molecular Biology CMB, and 'Department of Molecular Neurobiology, Karolinska Institute, Box 60400, Stockholm, S-10401, Sweden Communicated by B.Vennstrom

Thyroid hormones are essential for correct brain development, and since vertebrates express two thyroid hormone receptor genes (TRa and /3), we investigated TR gene expression during chick brain ontogenesis. In situ hybridization analyses showed that TRa mRNA was widely expressed from early embryonic stages, whereas TR,B was sharply induced after embryonic day 19 (E19), coinciding with the known hormone-sensitive period. Differential expression of TR mRNAs was striking in the cerebellum: TR,8 mRNA was induced in white matter and granule cells after the migratory phase, suggesting a main TR/ function in late, hormone-dependent glial and neuronal maturation. In contrast, TRa mRNA was expressed in the earlier proliferating and migrating granule cells, and in the more mature granular and Purkinje cell layers after hatching, indicating a role for TRa in both immature and mature neural cells. Surprisingly, both TR genes were expressed in early cerebellar outgrowth at E9, before known hormone requirements, with TR3 mRNA restricted to the ventricular epithelium of the metencephalon and TRa expressed in migrating cells and the early granular layer. The results implicate TRs with distinct functions in the early embryonic brain as well as in the late phase of hormone requirement. Key words: brain development/c-erbA/thyroid hormone receptors

Results

Introduction There has been a long-standing interest in the critical role of thyroid hormones (tri-iodothyronine, T3; thyroxine, T4) in development of the central nervous system (Schwartz, 1983). In man, the mental and physical retardation of cretinism has been associated with loss of thyroid gland function since last century (Osler, 1898; McCarrison, 1908). Also, experimental hypothyroidism in the rat and chick embryo results in impaired brain development (Dussault and Ruel, 1987; Bouvet et al., 1987). Various developmental abnormalities have been described in these animal models, particularly in the cerebellum and other regions which develop later during the known hormone-sensitive period. Thyroid hormone requirement has been implicated in neuronal and glial developmental processes including cell migration, myelination and synaptogenesis (Legrand, 1984). Earlier studies demonstrated increased T3 binding in brain during the hormone-dependent developmental period ©O Oxford University

(Schwartz and Oppenheimer, 1978; Perez-Castillo et al., 1985). Recently, the structure of thyroid hormone receptors (TRs) has been revealed by cDNA cloning, showing them to belong to the family of nuclear hormone receptors which act as ligand-responsive transcription factors (Sap et al., 1986; Weinberger et al., 1986; review: Green and Chambon, 1988). Various forms of TRs are produced from two genes (a and ,B) in the chick, rat and man (Benbrook and Pfahl, 1987; Murray et al., 1988; Hodin et al., 1989; Forrest et al., 1990). TRoe and : bind T3 with similar affinity, but amino acid differences in their DNA binding regions and variation in their N-termini suggest potential differences in trans-activating functions (Murray et al., 1988; Forrest et al., 1990), a view supported by differential expression of TRa and / genes in various tissues in vertebrate development (Forrest et al., 1990; Baker and Tata, 1990). Notably, in the chick and rat brain, TR/i mRNA undergoes dramatic induction late in development whereas TRat is expressed from early embryonic stages (Forrest et al., 1990; Strait et al., 1990). However, despite many descriptions of morphological defects in the developing brain in altered thyroid states, little is known of specific target cells for hormone action nor of the role of the different receptors. We have now identified by in situ hybridization analysis of TRa and ,B mRNAs, specific areas and cell types in the developing chick brain which are likely to be directly responsive to hormone. The results show striking differences in the timing and cell specificity of expression of TRca and ,B genes, especially in the proliferating and migrating cells of the cerebellum, implicating TRa and / with distinct late and early functions in brain development.

Press

In situ hybridization analysis of expression of thyroid hormone receptor a and ,d genes We have previously shown by Northern blot analysis that in chick brain development TRcx mRNA was present from early stages (embryonic day 4) and its levels increased 2-fold throughout development, whereas TR,3 mRNA was scarcely detectable before embryonic day 19 but was sharply induced upon hatching. To study this in detail we made antisense oligonucleotide probes specific for TRa and /3 mRNAs and a TR,B sense oligonucleotide as a control probe (C) for in situ hybridization analysis (Figure 1). We first established by Northern blot analysis that the probes specifically detected the expected mRNAs for TRa (4.5 and 3.0 kb) and /3 (7.0 kb) with characteristic tissue distribution, i.e. TRca mRNA was present in all tissues examined but TR,3 mRNA was restricted (e.g. absent in blood). No cross-hybridization of probes with different TR mRNAs was recorded. In the rat and human, C-terminal splice variants of TRca are produced which fail to bind T3 and which differ in transactivating properties (Izumo and Mahdavi, 1988; Koenig 269

D.Forrest et al.

Fig.

1.

Oligonucleotide probes specific

The structures of TRa

and

are

for chick

TRai and

mRNAs.

schematically represented, showing

putative zinc finger DNA binding domains and C-terminal binding regions; 3'UT, 3' untranslated sequence. Antisense oligonucleotides were made for regions of lower homology between

the

hormone

TRa and

at

marked

cDNA sequences A control

and

indicated underneath

as

probe, C,

was

made

as

by

the

arrows sense

sequence

antisense probe (see Materials and methods). complementary to the Northern blot analyses were performed with identical filters hybridized with 32 P-end-labelled oligonucleotides a, (3and C. Sizes of mRNAs detected are shown to the right of the respective filters (in kb). Lanes: P2 brain, mRNA from whole brain of day 2 post-hatching chick; other lanes, mRNA from tissues of embryos at days 16 (E16) and 19 (E19). Autoradiographic exposure times were 16 h (a) and 40 h (13 and C).

et

al., 1989). However, extensive analysis in development

of the brain and other tissues in the chick has failed to detect any obvious C-terminal variants of TRa

1990). Therefore in

situ

with

previously

we were

these

oligonucleotides

characterized

or

(Forrest

et

al.,

confident that mRNA detected would code

for the

forms of chicken TRa and

hybridization in situ were also optimized background and possible cross-hybridization.

Conditions for

minimize

Differential expression of TRa and brain

mRNAs

to

during

development

identify developing brain regions where TRat and were expressed, a series of brain sections throughout development were analysed in situ with 35 S-end-labelled and C as shown in Figure 2. All comparative probes ca, mRNA levels were performed with analyses of TRca and probes labelled to similar specific activities and employed equivalent hybridization and autoradiographic exposure To

mRNAs

conditions (see Materials and methods). TRa expression. TRa mRNA was found widely distributed 270

in many fore-, mid-, and hindbrain areas throughout development, even as early as embryonic day 5 (not all data shown). Expression was particularly evident in the cerebellum from early stages (E9); peak formation of the dense external granular cell layer (EGL) at E15 correlated with relatively strong TRat hybridization. After E15, high level expression was also detected in the internal granular layer (IGL) which forms upon inward migration of granule cells from the EGL. However, signal persisted in the residual EGL even after hatching (see below). White fibre tracts replace the cerebellar cavity from about E14 onwards in the chick (Pearson, 1972), and this area displayed considerably lower TRat hybridization during this period. Expression of TRa mRNA occurred during development of various layers of the telencephalon. Higher signals were detected in the accessory, dorsal and ventral hyperstriata and in the neostriatum, with slightly lower levels in the paleostriatal region (Figure 2A). Towards the mid-line of the caudal telencephalon, a region of higher TRa expression was detected in the hatched chick (P21, Figure 2A), apparently in the region of the hippocampal formation [although the hippocampus in the chick is significantly less developed than in mammals and is less well-characterized (Karten and Hodos, 1967; Pearson, 1972; Youngren and Phillips, 1978)]. At earlier developmental stages (E15) higher expression was also detected in this region. Relatively high TRa and ( mRNA levels have been reported to occur in the adult rat hippocampus (Bradley et al., 1989). In the developing optic lobe, higher TRa expression was detected in the external stratum opticum and internal stratum griseum centrale. The stratified expression pattern persisted in the hatched chick (Figure 2A, Table I). In the diencephalon, in the hatched chick we detected higher TRct mRNA levels in the epiphysis cerebri (pineal gland) and in the hypothalamus (Table I). Expression was generally lower in the brain stem. TR3 expression. Figure 2A demonstrates clearly the abrupt, late developmental elevation of TR3 mRNA in contrast to the earlier, more continuous expression of TRa mRNA. This was especially notable in the cerebellum where expression increased in two phases: (i) a small but clear increase over background at E19 with uniform expression in the white matter and IGL, and (ii) a sharp increase in the IGL, but not white matter, after hatching. Apart from in the cerebellum, when TR/ mRNA did become induced it was generally expressed in similar areas to those in which TRet nmRNA was already present, and it attained approximately comparable levels of expression to those of TRa (Table I). In the telencephalon, low level TR/, expression was apparent by E19, which further increased after hatching. However, by stage P4, differential hybridization was detected in the striatal layers, with somewhat higher levels in the accessory hyperstriatum and neostriatum and lower levels in the ventral hyperstriatum and paleostriatum augmentatum, contrasting with the more uniform expression of TRct in the telencephalon. In the hatched chick, TR,B mRNA was relatively highly expressed, similarly to TRa, in the region of hippocampal formation. In the optic lobe, TR/3 hybridization signal was detected by E19, which increased further in the hatched chick. Expression was more generally distributed compared with the stratified pattern seen with TRet. In the hatched chick, as with TRa, TR,B expression was also detected in the hypothalamus and pineal gland, both of which are organs

Differential expression of TRs in brain development

A

E9

E15

E19

P4

P21 B

A -TEL ) OL

P4 Control

-

CBL

A Fig. 2. In situ hybridization analysis of developmental expression of TRa and f3 mRNAs in the chick brain. (A) For each stage immediately adjacent para-sagittal sections were hybridized with probes a, f and C (a representative P4 stage control is shown) and exposed to autoradiographic film for 2 weeks. Hybridization signal detected as whiter areas. Ages (in days) of embryos (E9, 15 and 19) and hatched chicks (P4 and 21) are shown to the left (the chick hatches at E21). Abbreviations. CBL, cerebellum: MS, mesencephalon; OL, optic lobe; wm, white matter; ig, internal granular layer; eg, exteinal granular layer; ha, accessory hyperstriatum; hv, ventral hyperstriatum; n, neostriatum; pa, paleostriatum augmentatum. White scale bar = 5 mm. (B) Schematic depiction of sections, made along line A, in dorsal view of a representative E19 brain. Notes: (i) in part A, for P21, the section line was through a more mid-sagittal region in the forebrain; (ii) for E9 and E15, to retain truer structure, the brain was not removed from the head before sectioning.

thought to be involved in feedback regulation of thyroid hormone production in the chick (Wentworth and Ringer, 1986; Sharp et al., 1984).

Approximate levels of expression of TRa and ,B mRNAs in brain regions of the hatched chick are summarized in Table I. Background hybridization with the control probe was 271

D.Forrest et al. Table I. Expression of TRa and (3 mRNAs in brain regions of the hatched chick

Table II. Relative expression of TRa and ( mRNAs per cell in the developing cerebellar layers

Tissue/region

Region of cerebellum

Thyroid hormone receptor

Developmental stage E9 E15 E19

Probe

Telencephalon Accessory hyperstriatum Ventral hyperstriatum Neostriatum Paleostriatum Parolfactory lobe Olfactory bulb Hippocampal formation Diencephalon Hypothalamus Epiphysis cerebri Optic lobe Stratum opticum Stratum griseum et fibrosum superficiale Stratum griseum centrale Stratum album centrale Brain stem Cerebellum External granular layer Internal granular layer White matter

++ ++ + + ++ + +++ + +

+

++ + ++

-/+ -/+

Internal granular layer

+++

Purkinje cells

+ +

All tissuesa

control

-/ +

++ ++++

-/+

+ + +

++++ +

Expression levels of TRa and (3 mRNAs were determined from film autoradiographs of the hybridized sections shown in Figure 2A and other para-sagittal and coronal sections (not shown), on a scale ranging from not above background control (-) to relatively strong (++ + +).

minimal at all developmental stages examined, as seen in the representative P4 control section (Figure 2). Hybridization patterns for both TRa and ( were further confirmed by Northern blot analyses of RNA from dissected cerebellum, optic lobe and forebrain regions (not shown). It should be noted that the film autoradiograph signals seen in Figure 2 reflect both the hybridization signal per cell and also the cell density, which is significant for example, in the dense cerebellar granular layers and hippocampal region at later stages. Quantitation of signal per cell in the developing cerebellum is considered in detail below. Differential expression of TRar and , genes in the developing cerebellar layers In view of the striking differential expression of TRat and 3 mRNA in the developing cerebellum (Figure 2A) we examined the pattern in detail at the level of cellular resolution. Figure 3A shows dark field photographic analyses of expression of TRa and ,B mRNAs in an embryonic folium (E15) and in a more developed folium in the hatched chick (P3). As shown in Table II, upon quantitation as photographic silver grains/cell, intense TRa hybridization was detected at El5 in both the IGL and EGL, whereas TR3 signal was low in the IGL and not detected in the EGL. At stage P3, TRa mRNA expression was most intense in the cells of the IGL, although relatively strong signal persisted in the residual EGL. In the IGL at P3, TR( mRNA levels had increased to near those of TRa, and hybridization signals for both genes appeared to be uniformly distributed over the IGL cells when analyzed at higher magnification (not

shown). In the hatched chick, expression of TR3 mRNA was restricted mainly to the IGL and white matter and was 272

External granular layer

2.8 4.6 5.3 1.1

a

( a ( a (3 a

+

++ ++ +

Ventricular epithelium

1

7.7 1.1 9.2 1.9

1

4.6 0.7 6.2 2.1 5.7 1.7 1

P3

3.2 1.1 4.8 3.4 5.8 2.3 I

Embryonic and hatched chick brain sections were analyzed in situ with a, ( and control probes, and exposed to photographic emulsion as described in Figure 3. After development, sections were stained with cresyl violet to allow counting of cells and of silver grains located over cells. Values for TRa and (3 represent the mean number of grains/cell obtained from three or more different areas from at least two independently hybridized sections, and are normalized with respect to background hybridization of the control probe in equivalent sections (aindicates control signal levels assigned value 1 in all tissues). For each probe in the epithelium and granule cell layers, randomly chosen areas containing between 450 and 700 cells were analyzed, with actual control values between 0.4 and 0.7 mean number grains/cell. Purkinje cell values were based on analysis of between 24 and 39 cells for each probe with the actual control values between 4.5 and 4.8 mean number of grains/cell. Column spaces were left blank for developmental stages when particular cerebellar layers did not exist or were not sufficiently discernible.

not detected above background at any developmental stage in the EGL. Differences in TRae and 3 expression were also apparent in the Purkinje cell layer as shown in Figure 4 and Table II. TRa mRNA was detected at 3-fold higher levels than those of TRj in these large neurons in their final positions bordering the IGL at E19 and P3, whereas TRj hybridization was only slightly above background at either stage. -

Expression of TR genes in eady cerebellar morphogenesis Although TR( mRNA was detected only at very low basal levels in whole brain at stages before E19 (Forrest et al., 1990; Figure 2A), closer analysis of the early cerebellum at E9 surprisingly revealed a localized region of stronger expression in the epithelium and bordering mantle layer in the roof of the fourth ventricle (Figure 3B). Hybridization was not above background in the cells migrating outwardly from the epithelium nor in the primitive EGL. Upon quantitation as silver grains/cell (Table II), TR(3 mRNA was at 2-fold higher levels than those of TRa in the epithelium. In contrast, TRa mRNA was highly expressed in the migrating cells and was at 5-fold higher levels than TR(3 mRNA in the EGL at E9. The above-described patterns of TRca and ,B mRNA hybridization in the cerebellar layers throughout development were consistently seen in additional sections in other parasagittal and transverse planes (not shown). -

Discussion Our results demonstrate striking contrasts in expression of TRct and ( genes in brain development and point to the

Differential expression of TRs in brain development

A

tI

EG

E15

I

IG 2501J

&

P3

IG

WM

300-.

B EG

111 .or

VE

300i;

Fig. 3. Expression of TRca and ,B mRNAs in the developing cerebellum (A) Left hand panels for histological identification, show bright field views of para-sagittal sections of a cerebellar folium in a 15 day embryo (E15) and 3 day hatched chick (P3) (cresyl violet stain). Dark field photomicrographs shown on the right under a, ,B and C are at the same scale as the corresponding light field views. Hybridization appears as white signals. Immediately adjacent sections were hybridized with equivalent amounts of probes a, 1 and C, then dipped in photographic emulsion for 9 weeks exposure. White arrowheads indicate the external granular layer and black arrowheads the white matter (B) Left hand panel shows a cresyl violet stained para-sagittal section through the metencephalon at stage E9, and right hand panels corresponding dark field views of adjacent sections hybridized with probes ca, 13 and C. Ill and IV indicate the third and fourth ventricles respectively and white arrowheads the ventricular epithelium in the metencephalon roof. Abbreviations: EG, external granular layer; IG, internal granular layer; WM, white matter; VE, ventricular epithelium.

subtlety of mechanisms underlying the responses to thyroid hormones. In addition, the different kinetics of induction of TRca and ,B mRNAs indicate distinct temporal functions for TRa and in brain development. Study of hypothyroidism in man and animals has demonstrated a later developmental period during which thyroid hormones are essential for glial and neuronal maturation (Legrand, 1984). Our present results strongly implicate TR( with a key function during this period, which was particularly obvious in the cerebellum. The chick cerebellum evolves by outward migration of cells from the epithelium of the metencephalon roof as early as E8 to form the transient external granular cell layer (EGL) (Hanaway, 1967; Hallonet et al., 1990). Extensive proliferation of granule cells occurs until E15, at which time massive inward migration proceeds to form the internal granular layer (IGL). The granular cells meet outwardly migrating Purkinje and other cells to form by E19 the characteristic layers of the cerebellum, which is functional at hatching, since the newly hatched chick performs co-ordinated movement.

The sharp onset of TR,B mRNA expression in the cerebellar IGL occurs essentially after granule cell proliferation and migration are complete at E19, and correlates with the known hormone-sensitive period of synapse formation between the Purkinje cells and granule cell axons in the chick (Bouvet et al., 1987; Foelix and Oppenheim, 1974). This suggests a key role for TR,B in synaptogenesis as cerebellar neurons become active around the hatching period. Since the late expression of TR,B mRNA is mainly restricted to the IGL and white matter, we propose another TR(3 function in hormone-dependent myelination of axons by glial cells, in accord with the late increase in glial-specific T3 binding in chick brain (Haidar et al., 1983); myelination is necessary for onset of neuronal activity (Lemke, 1988). Similarly, thyroid hormones are also important in the rat in stimulating myelination markers in glia (Walters and Morrell, 1981; Clos et al., 1982) and in synapse formation (Vincent et al., 1982). Support for such a model awaits the characterization of T3 response elements of myelination genes and investigation of possible differences in their interaction with TRct and ,B.

273

D.Forrest et al.

L. 1 9 (I.

r.-i

EG.

PF

IG

2514:

Fig. 4. Comparison of expression of TRa and 13 mRNAs in cell layers of the cerebellum at stage E19. Adjacent para-sagittal sections were analyzed with TRa, 13 and control probes, exposed to photographic emulsion and stained with cresyl violet. Hybridization detected as photographic silver grains (black) over stained cells. Abbreviations: EG, external granular layer; IG, internal granular layer; P, Purkinje cell layer. Black arrowheads indicate individual Purkinje cell bodies.

During the known hormone-dependent period, TRat mRNA is comparatively highly expressed in the EGL and nascent IGL, whereas TR,B mRNA is not detected in the EGL. Since granule cell migration from E15 onwards is retarded in the hypothyroid chick (Bouvet et al., 1987), our results suggest a TRa-specific function in hormone-sensitive differentiation and migration of granule cells. Also, in both the rat and chick, hypothyroidism inhibits dendritic arborization and synapse formation of Purkinje cells (Vincent et al., 1982; Bouvet et al., 1987). Our analysis shows considerable expression of TRa mRNA but only low levels of TR,B, in the Purkinje cells at E19 and later, indicating that TRat may directly mediate late Purkinje cell maturation. More complex explanations might also invoke indirectly induced Purkinje cell differentiation by other T3-responsive cells, for example through synapse formation as mentioned above. A recent study limited only to the adult rat brain reported complete absence of TR,8 mRNA in the cerebellum and absence of TRa mRNA in Purkinje cells (Bradley et al., 1989), which may reflect species differences in T3 response mechanisms since such differences are already known between rat and chick brain in expression of TRa splice variants (Mitsuhashi et al., 1988; Forrest et al., 1990). Alternatively, our data suggest that a detailed developmental study of TR expression in rat brain is necessary before drawing this conclusion. It is intriguing that both TRat and ( genes are also expressed in the early brain, preceding the known period of thyroid hormone requirement. Differential expression is clearly seen in early cerebellar outgrowth at E9 where very localized expression of TR(3 mRNA in the epithelium of the metencephalic roof suggests a possible role for TR( in initiation of cerebellar morphogenesis. In contrast, TRca mRNA expression in outwardly migrating cells and in the primitive EGL at E9 indicates that TRa is important in early 274

migration and proliferation of granule cells as well as in the later phase discussed above. Developmental regulation of target gene networks may be facilitated by differential expression of TRa and (. Under some experimental conditions both TRa and (3 can individually activate transcription through a common response element (Koenig et al., 1989; M.Sjoberg, D.Forrest and B.Vennstrom, unpublished). However, we are investigating the specificity of DNA sequence recognition and possible cooperative or competitive interactions by different TRs, since co-expression of TRa and ,3 genes is apparent in some areas, for example, in the cerebellar IGL and some telencephalic regions in the hatched chick. Likewise, modulation of TR activity by retinoic acid receptors through recognition of common target genes (Graupner et al., 1989) or heterodimer formation (Glass et al., 1989), and involvement of other cell-specific factors (Bonde and Privalsky, 1990) also merits further study. Furthermore, TRs may suppress or activate target gene transcription in the respective absence or presence of T3 (Damm et al., 1989) suggesting another level of control of TR activity in the early embryo when thyroid hormone availability may be limited (Wentworth and Ringer, 1986). Thus, widespread early expression of TRa mRNA could reflect a more general role for TRca, perhaps as a T33-independent repressor in immature neural cells. Our present results emphasize the importance of TRj in the known period of thyroid hormone requirement in late brain development. In man, the TR(3 gene has also been associated with the neurological and growth abnormalities of the generalized thyroid hormone resistance syndrome (Usala et al., 1988). Surprisingly, we also indentified early embryonic expression of TRa and ,B genes. Moreover, early TR(3 expression is very restricted, so far being characterized only in the cerebellum and developing eye (Forrest et al., 1990; D.Forrest, M.Sjoberg, B.Vennstrom, unpublished data). This implicates TR,B with very specific functions in the early developing nervous system, as well as a key role in the later period of hormone requirement, and thus broadens the perspective in which the functions of TRs have previously been considered.

Materials and methods Preparation and analysis of RNA Poly(A)-selected RNA was prepared from embryonic and hatched chick tissues and 5 jsg aliquots analysed by Northern blot hybridization as described (Vennstrom and Bishop, 1982; Forrest et al., 1990), with the following modification: oligonucleotide probes were 32P-end-labelled using polynucleotide kinase and hybridization was in 10% formamide, 5 xSSC, 5 x Denhardt's solution, 20 mM phosphate buffer pH 7.5 at 43°C. Filters were then washed with increasing stringency to determine optimum conditions for probes a, a and C (0.1 xSSC at 560C). Oligonucleotide probes Antisense oligonucleotides were synthesized according to published chicken TRa and 1 cDNA sequences: a (49mer) nucleotides 432 -480 in Sap et al. (1986); 13 (47mer), nucleotides 1 179-1225 in Forrest et al. (1990). The 47mer control probe, C, was the complementary sense sequence to the 1 probe. Other oligonucleotides from different regions of cTRae and 13 were also tested as probes by Northern blot analysis, but were not sufficiently specific to be useful. Oligonucleotides were 3'-end-labelled with [a35S] dATP using terminal deoxynucleotidyl transferase to specific activities of -8 x 108 c.p.m./yg and purified for in situ hybridization use as described (Hallbook et al., 1990).

Differential expression of TRs in brain development Preparation of tissue sections and in situ hybridization analysis Brains were dissected from brown leghorn chicks and embryos and frozen on dry ice for cryostat sectioning. Sections were 10 ym thick for embryos up to E15 and 14 Am thick for older embryos and hatched chicks. Sections were mounted on poly-L-lysine-treated slides, fixed and hybridized as described (Hallbook et al., 1990). Sections were hybridized with equivalent amounts of probes ce, ,3 or C (2 x 106 c.p.m.) in 150 pA of hybridization solution per slide in a humidified chamber. After hybridization, sections were washed as follows (optimum conditions were first determined with a range of wash stringencies): 3 brief rinses in 0.5 xSSC, 14 mM /3-mercaptoethanol at room temperature, then 3 x 15 min in 0.5 x SSC, 14 mM ,B-mercaptoethanol, 0.1 % N-lauryl sarcosine at 55°C, a further 15 min in 0.3 xSSC including 03mercaptoethanol and N-lauryl sarcosine and finally a 30 min rinse in only 0.3 x SSC at room temperature. Sections were dried and were first exposed to Amersham ,3-max autoradiographic film for 2 weeks, then dipped in Kodak NTB-2 photographic emulsion and exposed for 9 weeks at -20°C, before developing and counter-staining with cresyl violet for photomicroscopic examination.

Acknowledgements We are grateful to Drs Robert Elde, Marc Hallonet and Tomas Hokfelt for valuable discussion and advice, Drs Ted Ebendal and Johan Thyberg for laboratory facilities and Gunnel Jonsson for secretarial assistance. We thank Dr Robert Elde for critical reading of the manuscript. D.F., F.H. and H.P. were supported by the Swedish Natural Science Council and B.V. by the Beijer Foundation. The work was also supported by the Swedish Cancer Society and the Knut and Alice Wallenberg Foundation.

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